Characterization of the biochemical activity and tumor-promoting role of the dual protein methyltransferase METL-13/METTL13 in Caenorhabditis elegans

The methyltransferase-like protein 13 (METTL13) methylates the eukaryotic elongation factor 1 alpha (eEF1A) on two locations: the N-terminal amino group and lysine 55. The absence of this methylation leads to reduced protein synthesis and cell proliferation in human cancer cells. Previous studies showed that METTL13 is dispensable in non-transformed cells, making it potentially interesting for cancer therapy. However, METTL13 has not been examined yet in whole animals. Here, we used the nematode Caenorhabditis elegans as a simple model to assess the functions of METTL13. Using methyltransferase assays and mass spectrometry, we show that the C. elegans METTL13 (METL-13) methylates eEF1A (EEF-1A) in the same way as the human protein. Crucially, the cancer-promoting role of METL-13 is also conserved and depends on the methylation of EEF-1A, like in human cells. At the same time, METL-13 appears dispensable for animal growth, development, and stress responses. This makes C. elegans a convenient whole-animal model for studying METL13-dependent carcinogenesis without the complications of interfering with essential wild-type functions.

To find out if nematode METL-13 has similar enzymatic activities as its human counterpart, we expressed and purified from E. coli two N-terminally hexahistidine (His 6 ) tagged fragments of METL-13 encompassing its individual MTase domains (Fig 1A). These METL-13 fragments were tested in an in vitro assay to monitor their putative MTase activities, using recombinant human (Hs) eEF1A as the protein substrate and [ 3 H]-AdoMet, as methyl donor, allowing the detection of [ 3 H]-methylated eEF1A by fluorography as a distinct band on SDS-PAGE. We decided to use the recombinant Hs eEF1A as the substrate based on its high homology to the nematode ortholog and nearly identical sequence surrounding the putative METL-13 target sites (S1A Fig in S1 File). We observed that the C-terminal domain of METL-13 could methylate eEF1A bearing a C-terminal His-tag, but not when the His-tag was present on the N-terminus, suggesting that the methylation occurs at the N-terminus of eEF1A (Fig  2A). Indeed, mutating G2 to I (G2I) abolished the methylation of eEF1A, whereas mutating K55 to R (K55R) had little effect (Fig 2A). In contrast, the N-terminal domain of METL-13 methylated Hs eEF1A, regardless of the position of the His-tag or the G2I substitution ( Fig  2B). Most importantly, the methylation was abolished by the K55R substitution, pointing to K55 of eEF1A as the key methylation site (Fig 2B). These experiments show that the enzymatic activities of individual domains of METL-13 match the established activities of human METTL13, i.e., its N-terminal domain targets K55, whereas the C-terminal domain targets G2 of eEF1A.
To demonstrate that METL-13 functions as a MTase in vivo, we used a previously uncharacterized mutant strain, carrying a homozygous deletion in the metl-13 locus. This mutation, metl-13(tm6870), causes a frameshift at the end of exon 3, leading to a premature stop codon (S1B Fig in S1 File). Nonsense-mediated mRNA decay (NMD) is expected to degrade the resulting mRNA and, indeed, the levels of metl-13 mRNA were significantly lower in the metl-13(tm6870) strain than in wild-type (wt) (Fig 2C). Moreover, any residual METL-13 protein would lack part of the N-terminal MTase domain and the whole C-terminal MTase domain. Thus, metl-13(tm6870) animals should express no functional METL-13 protein. To confirm that the metl-13(tm6870) strain is devoid of METL-13 activity, we analyzed the methylation status of G2 and K55 of the endogenous EEF-1A/eEF1A in metl-13(tm6890) and wild-type animals. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found that G2 on EEF-1A was fully trimethylated and K55 was virtually fully (99.6%) dimethylated in wild-type animals (Fig 2D and 2E). In contrast, both residues were completely unmethylated in metl-13(tm6870) mutants (Fig 2D and 2E). Thus, METL-13 is responsible for the G2 and K55 methylation of EEF-1A in vivo.
Finally, using surface sensing of translation (SUnSET) [29,30], we asked whether the loss of METL-13 impacts global protein synthesis. However, we observed no obvious difference between the wild type and metl-13(tm6870) mutants (Fig 3B). Concluding, the biochemical

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The C. elegans model for METL-13-dependent tumorigenesis activity of C. elegans METL-13 and human METTL13 is conserved. METTL13/METL-13 targets eEF1A/EEF-1A at two independent sites, introducing dimethylation at K55 and trimethylation at G2. However, at standard growth conditions, these modifications appear to have no obvious impact on global translation.

The K55 methylation of EEF-1A increases the penetrance of germline tumors
In humans, increased levels of METTL13 and eEF1A K55me2 correlate with poor survival of patients with lung or pancreatic cancer [25]. Furthermore, the ablation of METTL13 reduces proliferation and global protein synthesis in cancer cells, while non-transformed cells appear unaffected [25,26]. Since the biochemical function of METL-13 is conserved in C. elegans, we tested whether it also plays a role in tumorigenesis. Many C. elegans factors function as germline tumor suppressors [31]. Among them is GLD-1, a maxi-KH/STAR RNA-binding protein functioning as a translational repressor [32][33][34][35]. Another example is PRO-1, a conserved WDrepeat protein implicated in rRNA processing [36]. Without these factors, germ cells develop into highly penetrant tumors, although found in different gonadal regions (Fig 4A).
Therefore, to evaluate the role of METL-13 in tumorigenesis, we knocked down gld-1 by RNAi in either wild-type animals or metl-13(tm6870) mutants. Upon depletion of GLD-1, germ cells that have entered meiosis revert to the mitotic cycle, resulting in an ectopic mass of proliferative cells that we refer to as a 'fully proliferative' phenotype. In the wild type, most gonads displayed large tumors in the proximal/medial regions, as expected; these tumors consisted of small, undifferentiated cells (Fig 4B). In the metl-13(tm6870) mutants, however, many of the tumorous gonads contained enlarged cells reminiscent of differentiating oocytes, we refer to this gonad phenotype as 'proliferative with enlarged cells' (Fig 4B and S2A Fig in  S1 File). Importantly, we observed a similar increase in the differentiating cells in the eef-1A.1

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The C. elegans model for METL-13-dependent tumorigenesis While the gonads of gld-1(q485) mutants display large proximal germline tumors, the gonads of pro-1(na48) mutants grown at the restrictive temperature (25˚C) contain smaller tumors located proximal to the gametes. B. DIC micrographs of adult wt animals subjected to gld-1 RNAi. The gonads are outlined. Three phenotypes were observed after gld-1 RNAi, we refer to these gonads as 'fully proliferative', 'proliferative with enlarged cells' (black arrowheads indicate enlarged cells), and 'wt-like' respectively. Scale bar = 20 μm. Quantification of the germline phenotypes observed after gld-1 RNAi in wt, metl-13(tm6870), and eef-1A.1(syb2837); eef-1A.2(syb2785) animals show that the predominant phenotype is 'fully proliferative' in wt and 'proliferative with enlarged cells' in metl-13(tm6870) and eef-1A.1(syb2837; eef-1A.2(syb2785) animals. The quantification shows the average phenotype distribution from three biological replicates, 70 germlines were assessed per biological replicate. C. DIC micrographs of pro-1(na48) mutants (grown at the restrictive temperature, 25˚C). The gonads are outlined. Three phenotypes were observed: we refer to seemingly normal germlines as 'wt-like', germlines containing proliferating cells proximal to the gametes as 'proximal tumor' (indicated by a white arrow), and germlines lacking a proximal tumor that instead contain abnormal oocytes as 'abnormal oocytes' (indicated by black arrowheads). Scale (syb2837); eef-1A.2(syb2785) double mutants (Fig 4B and S2A Fig in S1 File). These differences were not caused by less efficient RNAi in the absence of METL-13-mediated methylation, as RNAi against another target was as efficient in the metl-13(tm6870) or eef-1A.1 (syb2837); eef-1A.2(syb2785) mutants as in the wild-type (S3A Fig in S1 File). These results suggest that the tumors are less penetrant without the K55 methylation of EEF-1A. To confirm this finding in another tumorous background, we RNAi-depleted metl-13 in temperature-sensitive pro-1(na48) mutants. At the restrictive temperature (25˚C), most of the pro-1(na48) gonads displayed proximal tumors, while a minority looked superficially wild-type or contained abnormal oocytes (Fig 4C). However, the numbers of gonads with proximal tumors significantly decreased upon metl-13 RNAi, and the fraction of wt-like gonads/gonads containing abnormal oocytes increased (Fig 4C and S2B Fig in S1 File). Concluding, our results suggest that METL-13-mediated methylation of EEF-1A enhances tumorigenesis in the nematodes, which is reminiscent of the tumor-promoting role of human METTL13.

METL-13 is non-essential for animal growth and development
To analyze the potential function of METL-13 in wild-type animals, we singled synchronized L4 wild-type or metl-13(tm6870) animals to individual plates, allowed them to grow, and counted their offspring. Since the speed of C. elegans development and the offspring size depend on temperature [37], these experiments were performed at 15˚C, 20˚C, and 25˚C ( Fig  5A). At all tested temperatures, we observed no difference in the offspring or developmental speed between the wild-type and mutant animals (Fig 5B).
Since tumorigenesis is linked to increased protein synthesis, we also examined animals cultivated on bacterial diet enhancing growth and development. In contrast to a standard diet of the E. coli OP50 strain, animals fed the E. coli HB101 strain develop faster and grow larger [38][39][40][41]. This is due to an increased cell size rather than cell number and reflects the increased protein content of HB101-fed animals [40]. To further accelerate growth and thus the demand for protein synthesis, the animals were cultured at 25˚C [37]. As anticipated, wild-type animals that were fed HB101 bacteria developed faster and grew bigger. However, there was no difference between the wild-type and metl-13(tm6870) mutants (Fig 5C and 5D). Thus METL-13 appears dispensable for wild-type growth and development, even under conditions requiring increased protein synthesis.
Finally, when comparing the lifespan of animals with or without functional METL-13, we noticed a small but significant difference, with the metl-13(tm6870) mutants dying a little faster than the wild-type (Fig 6). However, the lifespan of eef-1A.1(syb2837); eef-1A.2(syb2785) double mutants was not different from the wild-type (Fig 6), suggesting that the longevity-related role of METL-13 is unrelated to the K55 methylation of EEF-1A.

Discussion
Here, we have studied the previously uncharacterized C. elegans orthologue METL-13. The biochemical function of METL-13 is remarkably conserved, with the dual methyltransferase bar = 20 μm. Quantification of the germline phenotypes observed after mock or metl-13 RNAi show a decrease in the 'proximal tumor' phenotype and an increase in the 'abnormal oocytes' and 'wt-like' phenotypes when metl-13 is knocked-down. The quantification shows the average phenotype distribution from three biological replicates, 70 germlines were assessed per biological replicate. https://doi.org/10.1371/journal.pone.0287558.g004

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The C. elegans model for METL-13-dependent tumorigenesis being responsible for the same methylation marks in vivo on the nematode EEF-1A/eEF1A as in humans.
There is much interest in METTL13 due to its apparent role in cancer. METTL13 and eEF1A K55me2 are upregulated in a plethora of human cancers [25][26][27][28]. Moreover, targeting METTL13 or the downstream eEF1A K55 inhibits neoplastic growth in human cell lines, as well

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The C. elegans model for METL-13-dependent tumorigenesis as in xenograft and PDAC mouse models [25,26]. Remarkably, the depletion of METL-13 has a similar inhibitory effect on the C. elegans tumors, as the severity of genetically induced germline tumors declined in the absence of METL-13 or EEF-1A K55me2 . We note that inhibiting METL-13 or EEF-1A K55me2 did not suppress tumors completely. Also, the partial suppression was no longer observed when we used the gld-1(q485) null mutants instead of gld-1 RNAi (S3B Fig in S1 File). Thus, the loss of EEF-1A K55me2 reduces but does not abolish cancer, at least in the tested tumorous genetic backgrounds.
So how does eEF1A K55me2 impact cancer? The methylation of K55 upregulates the GTPase activity of eEF1A, which may cause increased protein synthesis needed for tumorigenic growth [25]. The elongation factor eEF1A is known to be present in excess compared to other components of the translation machinery. This suggests that eEF1A is unlikely to be rate-limiting in protein synthesis and that its non-canonical functions might instead play a role in cancer [42,43]. Alternatively, translation capacity may be in surplus under normal conditions but becomes limiting in cancer. This is consistent with the finding that eIF4E haploinsufficient mice develop normally, with wild-type levels of protein synthesis, but are more resistant to oncogenic transformation [44]. While the etiology of human and C. elegans tumors is likely different, it is intriguing that mutating various C. elegans RNA-binding proteins, which like GLD-1 function to repress translation, leads to germline tumors [31]. Thus, reducing translational output, by removing METL-13, could explain the partial suppression of the tumorous proliferation observed upon the depletion of GLD-1. Whether the same applies to pro-1 tumors is less clear, as their etiology is less well understood [36].
Cancer treatments typically involve the inhibition of essential proteins like cell cycle regulators and signaling molecules. This is why chemotherapy often causes serious adverse effects in cancer patients [45][46][47]. The METTL13-mediated methylation of eEF1A could be inhibited by small molecules. However, METTL13 knock-outs have thus far only been studied in cell lines and specific tissues [25,26]. Our whole-animal study suggests that a complete removal of METL-13 or EEF1-A K55me2 has no obvious impact on wild-type growth and development. This does not preclude potential functions under specific conditions or contexts. Curiously, METL-13 appears to play a limited but significant role in longevity. However, animals in which EEF-1A is no longer methylated on K55 do not display this longevity defect. Thus, this effect could depend on METTL13-mediated methylation of the G2 residue on EEF-1A, the methylation of another substrate, or an additional function of METTL13. In summary, our studies establish a simple model for METL-13-assisted tumorigenesis and endorse the eEF1A K55 modification as an attractive target in cancer therapy, whose inhibition could help

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The C. elegans model for METL-13-dependent tumorigenesis reduce cancer growth without the side effects associated with traditional drugs used in chemotherapy.

C. elegans culture and generation of mutant strains
Animals were maintained using standard procedures and grown at 20˚C unless stated otherwise [41]. Information on the strains used in this study are reported in Table 1. The metl-13 (tm6870) strain was outcrossed thrice with wild-type males to reduce background mutations. The eef-1A.1(syb2837) and eef-1A.2(syb2785) strains were generated by SunyBiotech using CRISPR-Cas9 technology and outcrossed thrice with wild-type males before crossing them together to generate the double mutant, which was again outcrossed three-times with wildtype males to reduce background mutations. To synchronize animals, embryos were extracted from gravid adults by treatment with a bleaching solution (30% (v/v) sodium hypochlorite (5% chlorine) reagent (ThermoFisher Scientific; 419550010), 750 mM KOH). The embryos were left to hatch overnight into arrested L1 larvae in the absence of food, at RT in M9 buffer (42 mM Na 2 HPO 4 , 22 mM KH 2 PO 4 , 86 mM NaCl, 1 mM MgSO 4 ). For RNAi experiments, synchronized L1s were grown at 25˚C to adults on RNAi-inducing NGM agar plates seeded with HT115 E. coli bacteria containing plasmids targeting genes of interest [48].

Cloning of METL-13
The ORFs encoding fragments of METL-13 protein (MT13-N: amino acids 1-417; MT13-C: amino acids 213-656), were amplified by PCR from C. elegans cDNA and cloned between NdeI and NotI sites into pET28a plasmid (Novagen), using In-Fusion1 HD Cloning Plus kit (Takara). The cloning primers are listed in Table 2. Sanger sequencing was used to verify all cloned constructs.

Expression and purification of recombinant proteins
Expression and purification of recombinant N-terminally His 6 -tagged C. elegans MT13-N and MT13-C proteins from E. coli was performed using Ni-NTA-agarose (Qiagen), following a

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The C. elegans model for METL-13-dependent tumorigenesis previously described protocol [49]. Cloning, mutagenesis and purification of recombinant human eEF1A1, containing either N-terminal or C-terminal His 6 -tag, was described previously [21,24]. Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Recombinant proteins were stored in Storage Buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 10% glycerol) in single use aliquots, kept at −20˚C.

In vitro methyltransferase assay using [ 3 H]AdoMet
MTase activity assay using C. elegans MT13-N or MT13-C as enzymes, human eEF1A1 as the substrate and [ 3 H]AdoMet (PerkinElmer) as the methyl donor, was performed similarly as previously described [24]. 10 μl reactions were set-up on ice in Storage Buffer, containing 1 μg of eEF1A1, 1 μg of MT13-N or MT13-C and 0.5 μCi of [ 3 H]AdoMet. Reaction mixtures were incubated for 1 h at 30˚C and analyzed by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membrane and stained with Ponceau S. Incorporation of tritium-labeled methyl groups into proteins was visualized by fluorography. All methyltransferase experiments were independently replicated at least two times.

RNA extraction
Pellets of 100 μl containing a mix of embryos, larvae and adults were prepared by harvesting the animals (grown on NGM agar plates with OP50 bacteria), adding 250 μl Trizol and snapfreezing in liquid nitrogen. Animals were lysed by 10 freeze-thaw cycles with liquid nitrogen and incubating at 37˚C respectively. Chloroform extraction was used by adding 50 μl chloroform and centrifugation at 12000 x g for 10 minutes. The aqueous phase was transferred into new microcentrifuge tubes and 1 volume of isopropanol was added and mixed well. The samples were centrifugated at 16100 x g for 20 minutes at 4˚C, after which the RNA pellet was washed with 80% ethanol and air-dried before being resuspended in nuclease-free water. The quality of the RNA was assessed using NanoDrop Spectrophotometer.

Enrichment of EEF-1A from nematode extracts
Pellets containing a mix of embryos, larvae and adults were prepared by collecting the animals (grown on NGM agar plates with E. coli OP50 bacteria) in M9 buffer and washing thrice before removing all of the buffer and snap-freezing in liquid nitrogen. Extracts were prepared by grinding the pellet with a mortar and pestle in the presence of liquid nitrogen and dissolving in Lysis Buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% Triton X-100, 5% glycerol (w/vol), 5 mg/ml cOmplete Proteinase Inhibitor Tablets (Roche, 11697498001), 1% P8340 (Sigma)). Extracts were cleared by centrifugation at 16100 x g for 5 minutes at 4˚C. EEF-1A protein was enriched from the C. elegans extracts using a method previously employed for enrichment of eEF1A1 present in human cell extracts [21]. Extracts were loaded on Pierce Strong Cation Exchange (S) Spin Columns (Thermo Fisher Scientific, 90008) pre-equilibrated in Lysis Buffer, followed by extensive washing of the columns with Washing Buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5% glycerol (w/vol), 5 mg/ml cOmplete Proteinase Inhibitor Tablets (Roche, 11697498001), 1% P8340 (Sigma)). The retained material, including EEF-1A, was eluted from the columns with Elution Buffer (50 mM Tris-HCl pH 7.4, 400 mM NaCl, 5% glycerol (w/vol), 5 mg/ml cOmplete Proteinase Inhibitor Tablets (Roche, 11697498001), 1% P8340 (Sigma)).

Mass spectrometry
Mass spectrometry analysis was essentially performed as previously described [24]. In brief, protein samples enriched for EEF-1A (obtained as described above) were resolved by SDS-PAGE and stained with Coomassie Blue. A band corresponding to the mass of EEF-1A (~50 kDa) was excised from the gel and subjected to in-gel digestion using chymotrypsin or Glu-C, after which the resulting proteolytic fragments were desalted using 10 μl OMIX C18 micro-SPE pipette tips (Agilent, Santa Clara, CA, USA). The samples were analyzed by nanoLC-MS using either an Ultimate 3000 RSLCnano-UHPLC system connected to a Q Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) or a NanoElute-UHPLC coupled to a TimsTOF Pro mass spectrometer (Bruker Daltonics, Bremen, Germany). The LC-MS data were analyzed using Peaks Studio version 10.6 (Bioinformatics Solution Inc.), searching against the C. elegans protein sequence database from Swiss-Prot (4155 entries). The maximum number of variable modifications detected on a peptide was restricted to three, allowing the following variable modifications: methionine oxidation, cysteine propionamidation, methylation of lysine (mono, di, and tri), and methylation of the N-terminus (mono, di, and tri). MS/MS spectra of peptides containing the N-terminal G2 or K55 of EEF-1A were inspected for presence of methylated residues, followed by quantitation of G2 and K55 methylation status using PEAKS Studio. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD042540 and 10.6019/PXD042540 [50].

Western blot analysis
Pellets containing a mix of embryos, larvae and adults were prepared as described above. Lysates were prepared by grinding the pellet with a mortar and pestle in the presence of liquid nitrogen and dissolving in lysis buffer (

SUnSET assay
Synchronous arrested L1 larvae were obtained as described above. A total of 16000 L1 larvae were grown until the young adult stage on NGM plates seeded with E. coli OP50 bacteria. Animals were washed, grown and treated with puromycin as previously described [29]. For the Western blot analysis, three micrograms of total protein were used per well. Incorporation of puromycin into nascent peptides was measured by normalizing band intensity from monoclonal anti-puromycin (Merck, MABE343) antibody to anti-actin (Abcam, ab8227).

Microscopy
Animals were transferred into a drop of 10 mM levamisole on a 2% (w/v) agarose pad, covered with a cover slip and immediately imaged with a Zeiss AxioImager Z1 microscope. Images were acquired with an Axiocam MRm REV2 CCD camera using the Zen software (Zeiss). The acquired images were processed with Image J.

Development and fecundity assay
Synchronized arrested L1 larvae were plated on NGM agar plates with OP50 and grown at 25˚C for 24 hours to the L4 larval stage. Individual animals were picked over so that there is one single worm per NGM agar plate. The animals and their offspring were left to grow at 25˚C, 20˚C, or 15˚C. The parental animal was picked over to a new plate every 24 hours and the offspring was counted and removed from the plate when they reached the L4 stage.

Body area measurements
Synchronous arrested L1 larvae were obtained as described above. A minimum of 50 animals were grown at 25˚C until 1-day old adults on NGM plates seeded with either E. coli OP50 or HB101 bacteria. Micrographs depicting the whole animal were taken of a minimum of 15 worms for each strain and each food-source condition per biological replicate. Body area of the animals in square pixels was measured with Image J.

Egg-to-egg assay
Four adult animals were picked on an NGM plate seeded with either E. coli OP50 or E. coli HB101 bacteria to lay eggs at 25˚C for 2 hours before being removed. The eggs were grown at 25˚C into the L4 stage before separating a single worm per plate for a total of 20 worms for each strain and food-source condition per biological replicate. The animals were kept at 25˚C and checked every hour after entering adulthood to mark the time at which they start laying their first egg.

Lifespan assay
Synchronous arrested L1 larvae were obtained as described above. A minimum of 100 L1 larvae were plated out per NGM agar plate and left to grow until the young adult stage at 20˚C. Dead animals were counted and removed from the plate every 48 hours. The population of animals was moved to a new plate every 24 hours during fertile adulthood to omit the offspring from the assay.
Supporting information S1 File. Contains all the supporting figures.